Non-targeted effects of radiation: a personal perspective on the role of exosomes in an evolving paradigm

Abstract

Purpose

Radiation-induced non-targeted effects (NTE) have implications in a variety of areas relevant to radiation biology. Here we evaluate the various cargo associated with exosomal signaling and how they work synergistically to initiate and propagate the non-targeted effects including genomic instability and bystander effects.

Conclusions

Extra cellular vesicles, in particular exosomes, have been shown to carry bystander signals. Exosome cargo may contain nucleic acids, both DNA and RNA, as well as proteins, lipids, and metabolites. These cargo molecules have all been considered as potential mediators of NTE. A review of current literature shows mounting evidence of a role for ionizing radiation in modulating both the numbers of exosomes released from affected cells as well as the content of their cargo, and that these exosomes can instigate functional changes in recipient cells. However, there are significant gaps in our understanding, particularly regarding modified exosome cargo after radiation exposure and the functional changes induced in recipient cells.

Keywords:

• Ionizing radiation
• exosomes
• bystander effects
• genomic instability
• intercellular communication
• extracellular vesicles

Non-targeted effects (NTE): a historical perspective

In the early 1990s, analysis of experimental data on the biological effects of radiation started to raise doubts about results that challenged the conventional paradigm that genetic alterations are restricted to direct DNA damage. These observations ushered a paradigm shift toward the NTE of radiation whereby cells that are not exposed to ionizing radiation show responses similar to those observed with direct radiation exposure. The NTE paradigm was first demonstrated in the descendants of irradiated cells which is now referred to as radiation-induced genomic instability (GI). Moreover, similar effects were soon observed in un-irradiated cells that were either in contact or receive signals from irradiated cells, a phenomenon currently known as radiation-induced bystander effect (RIBE). I am pleased that I contributed to the first pioneering manuscript (Kadhim et al. 1992) on this paradigm shift while working at the Medical Research Council (MRC) Radiobiology Unit, UK. We observed enhanced frequencies of non-clonal chromosomal aberrations in clonal descendants of mouse hematopoietic stem cells examined 12–14 generations following alpha radiation exposure. This was then extended in Kadhim et al. (1994) by showing these phenomena occur in human hematopoietic stem cells (Kadhim et al. 1994). We also discovered a genetic factor contributed to the induction of these effects, and thereby provided more evidence supporting the hypothesis that the instability phenotype is determined by genetic predisposition.

The transmission of chromosomal instability in vivo was reported by our MRC group (Watson et al. 1996), in mouse hematopoietic cells. Using the same system, Clutton et al. (1996) from the MRC, demonstrated that oxidative stress and resultant injury might generate a biochemical mechanism for both triggering and sustaining many endpoints of genomic instability (Clutton et al. 1996). In collaboration with the Biophysics group, we provided evidence of the first link between radiation-induced GI/chromosomal instability and bystander effects (BEs). This was established by a direct experiment where we used a grid technique in the hematopoietic cell system (Lorimore et al. 1998). Results from these experiments demonstrated that majority of the progeny exhibiting GI were from the un-irradiated cell population, leading to the conclusion that induction of this damage was caused by intercellular communication i.e. a bystander mechanism.

This scientific journey gave me the privilege to work and interact with many great national and international experts in the field of Radiation Biology to whom I am grateful for helping to refine my knowledge and research expertise. I also benefited by having the chance to supervise several postgraduate students, and to recruit several young research assistants as well as post-doctoral scientists many of whom continued to work in the field of radiation biology. Of course, this would not have been possible without generous funding and support awarded to my research group including: research funds from the US Department of Energy (DOE), several EU Framework projects, RADINSTAB, NOTE, DoReMi, and CONCERT. Also, many thanks to “The Dunhill Medical Trust,” and last but not least the early support from the MRC and the support of Oxford Brookes University.

The present paper reviews the historical background of evolution of NTE, it covers the contributions of other groups/scientists that shaped the current knowledge of non-targeted effects of radiation, and their importance in the health effects of exposure to ionizing radiation. The paper also highlights some current and future challenging research directions that help assess and improve our understanding of the mechanism of NTE and its possible implications for treatment, diagnosis, and safer occupational exposures.

Introduction

Radiation induced non-targeted effects

Cellular damage following exposure to ionizing radiation such as chromosomal aberrations and mutations can ultimately lead to cancer. In the past, this damage was linked to the direct absorption of energy in DNA and other cellular macromolecules, or to water radiolysis and subsequent free radical damage. However, toward the end of the last century, several additions collectively referred to as non-targeted effects (NTE) were made to radiation theory. Radiation-induced NTE were first studied in the 1990s with numerous publications reporting various effects. Since then, the key processes have been established (Table 1) as genomic instability (GI), BEs, abscopal effects (AE), and adaptive response (AR) although this is in some cases still disputed. NTE refer to pathways that cause damage to cells independent of the well-characterized DNA damage observed soon after irradiation. Radiation-induced genomic instability (RIGI) is a temporal phenomenon that can occur in the descendants of irradiated cells. On the other hand, radiation-induced BEs (RIBEs) arise in a spatial manner, that is, in cells that have not been exposed to IR but have communicated with cells that have been exposed to IR. In the body, the extent of cell signaling following irradiation is not just restricted to nearby cells but has been observed at distant sites. These are commonly referred to as abscopal effects (Barker et al. 2015).

Table 1. General definitions of non-targeted effects (NTE) of radiation.

NTE	Definition
NTE NTE of ionizing radiation NNNNNNTENNNNN	NTE are defined as responses that are expressed in the absence of direct ionizing radiation deposition in nuclear DNA. The majority of NTE are low dose effects and usually display non-linear dose-response relationships. NTE do not contradict “target theory” but contribute to a concept of an “expanding target” which is linked to underlying biological signaling triggered by physical dose deposition (Kadhim et al. 2013; Bright and Kadhim 2018)
Radiation induced genomic instability (RIGI)	RIGI is defined as a temporary or long-term increase in the rate of genetic changes in the genome of irradiated cells over many cell generations (Limoli 2017))
Radiation induced bystander effects (RIBE)	RIBE are radiation induced effects observed in un-irradiated cells that are in close proximity to irradiated cells as a result of communication with irradiated cells; either by secretion of soluble substances or signaling via gap junctions (Mothersill et al. 2018)
Abscopal effects (AE)	AE are transmitted between irradiated and un-irradiated tissues outside of the irradiated volume via systemic signaling (Pouget et al. 2018)
Adaptive response (AR)	AR is characterized by biological systems which are exposed to a small “priming” radiation dose to attenuate the effects of a larger challenging dose through activation of numerous signaling pathways (Calabrese and Baldwin 2001)

Although it has been suggested that NTE could have implications for cancer radiotherapy (as recently reviewed by Mothersill et al. 2019), here we explore two cellular manifestations of NTE, namely GI and BE. In particular, we focus on EVs, such as exosomes, as delivery vehicles for bystander signals and their impact on neighboring cells, which is relevant to radiation-induced NTE. Other more selective reviews of the ongoing research in this area have been published recently (for more details see: Kadhim et al. 2013; Desouky et al. 2015; Kadhim and Hill 2015; Mothersill and Seymour 2015; Burtt et al. 2016; Mavragani et al. 2017; Bright and Kadhim 2018; Yahyapour et al. 2018; Mothersill et al. 2019).

Genomic instability

GI refers to the process of a cell losing the ability to control and maintain the integrity of its genome (Figure 1). It is characterized by a genetically unstable phenotype that is transmitted to the progeny as seen by an increased acquisition of genetic alterations. The word “instability” came from early observations that the cellular aberrations observed in these cells are non-clonal in nature, an indication that new aberrations must arise in each cell generation through an ongoing mutational process (Kadhim et al. 1992). The types of aberrations include de novo chromosomal aberrations, higher levels of sister chromatid exchanges (Narayanan et al. 1997; Daino et al. 2002; Sowa Resat and Morgan 2004), changes in ploidy (Morgan 2003a, 2003b), micronuclei, transformation, gene amplification, gene mutation, reduced plating efficiency, lethal mutations (delayed reproductive cell death), and mini- and micro-satellite instabilities in vitro (Kadhim et al. 1992; Jamali and Trott 1996; Dubrova et al. 1998) and in vivo (Salomaa et al. 1998; Watson et al. 2001; Tanaka et al. 2008; Suman et al. 2017). GI is a hallmark of carcinogenesis and tumorigenic progression (Negrini et al. 2010; Hanahan and Weinberg 2011). The frequency of genomic instability in the progeny of irradiated cells is several orders of magnitude higher than would be expected for a single gene mutation, suggesting that a more intricate phenomenon may be underlying the phenotype (Kadhim et al. 1992; Kronenberg 1994; Morgan et al. 1996; Limoli et al. 1998; Morgan 2003a, 2003b; Averbeck 2010; Miller et al. 2010; Salomaa et al. 2010; Mothersill and Seymour 2012; Kadhim et al. 2013; Kadhim and Hill 2015; Burtt et al. 2016). There are several factors which influence the induction of genomic instability including radiation quality and dose, dose rate, genetic predisposition, and cell type (Kadhim et al. 2013; Kadhim and Hill 2015; Burtt et al. 2016; Elbakrawy et al. 2020).

Figure 1. Exposure to ionizing radiation can cause DNA damage to nuclear DNA. The cell can either repair any damage presents (blue cells) or mis-repair the damage fixing an aberration that is present in the progeny of that cell (pink cells). Alternatively, an ongoing process may occur in which new aberrations appear in some cells at a delayed time point.

The majority of studies indicates that GI and radiation dose is non-linear, but can be induced at extremely low doses such as traversal of a cell nucleus by a single alpha particle in a population of cells (Kadhim et al. 2001; Moore et al. 2005a).

A number of studies have attempted to identify the mechanisms behind radiation-induced GI. It is widely thought that GI is not caused by direct damage to DNA during irradiation. Mechanistically a number of observations have been linked to GI induction and include epigenetic alterations (Kovalchuk and Baulch 2008; Tamminga and Kovalchuk 2011; Mothersill and Seymour 2012; Merrifield and Kovalchuk 2013; Belli and Antonella Tabocchini 2020), persistent oxidative stress (Limoli and Giedzinski 2003; Snyder and Morgan 2003), and inflammatory-like processes (Lorimore and Wright 2003; Hamasaki et al. 2007; Mukherjee et al. 2014; Werner et al. 2015).

Once initiated, GI is likely to contribute to the evolution of a tumor and aids in its acquisition of new hallmarks of cancer (Pikor et al. 2013; Tubbs and Nussenzweig 2017). It is also proposed that GI is linked to other disease states such as neurodegeneration or even cataract formation (Chow and Herrup 2015; Hamada and Fujimichi 2015).

BEs

Radiation-induced BE are defined as radiation-like effects observed in un-irradiated cells as a consequence of cellular communication with irradiated cells (Figure 2). Communication can occur through gap junctions (protein channels that link adjacent cells) or molecular signals released in the extracellular milieu by irradiated cells (Hei et al. 2008). BE have been observed in a range of cell types for several biological endpoints, as a result of external beam or radionuclide exposures (Hei et al. 2008; Little et al. 2008; Blyth and Sykes 2011; Mothersill and Seymour 2012; Mothersill et al. 2018).

Figure 2. Exposure to irradiation can induce intercellular signaling with the appearance of radiation-like effects in un-irradiated cells. A number of factors such as reactive oxygen/nitrogen (ROS/RNS), cell free DNA (cfDNA) species, TNF-related apoptosis-inducing ligand (TRAIL) as well as a host of other proteins (various black shapes) have been proposed.

Experimental approaches have been discussed in detail (Table 1 in Widel 2016). The manifestations of BE share some similarity with GI indicating there could be commonality between the mechanism of induction. Manifestations include increased levels of mutations (Huo et al. 2001; Nagasawa et al. 2003), chromosomal aberrations (Lorimore et al. 1998; Nagasawa and Little 2002; Bowler et al. 2006), induction of micronuclei and sister chromatid exchanges, gene amplifications and mutations (Mettler and Upton 2008), induction of cellular senescence (Elbakrawy et al. 2020), phosphorylation of proteins such as ERK1/2, JNK and p38 (Little et al. 2002), gene expression changes (Azzam et al. 2002; Yang et al. 2005) changes in γH2AX foci indicating DNA damage (Hu et al. 2005; Sokolov et al. 2005; Yang et al. 2005), changes in cell proliferation, cell-cycle control, cell transformation, cell death, and apoptosis (Mothersill and Seymour 1997; Lewis et al. 2001; Sawant et al. 2001; Belyakov et al. 2002; Lyng et al. 2002; Nagasawa and Little 2002; Gerashchenko and Howell 2003; Zhu et al. 2005; Mothersill et al. 2006; Sokolov et al. 2007).

As also observed in GI, many bystander responses tend to saturate at low radiation doses regardless of LET (Seymour and Mothersill 2000; Little et al. 2002; Moore et al. 2005b). Furthermore, maximal effects can be observed by the lowest doses investigated (∼10 mGy) in some cellular systems (Nagasawa and Little 1992; Schettino et al. 2005; Kadhim et al. 2010). BE have also been documented when only cytoplasm is irradiated (Wu et al. 1999), with very low doses of low (Mothersill et al. 2002) or high-LET irradiation (Moore et al. 2005a; Bowler et al.2006; Lad et al.2019; Elbakrawy et al. 2020).

There is also substantial evidence for BE in vivo. Increased numbers of head tumors were reported in head-shielded mice whose bodies were irradiated demonstrating that BEs could be carcinogenic (Mancuso et al. 2013). Furthermore, in vivo BEs in non-irradiated fish receiving signals from irradiated individuals have been demonstrated (Mothersill et al. 2012). Also, Rusin et al. observed the effect of gamma radiation on generation of bystander signals from three earthworm species irradiated in vivo. The study showed evidence of bystander signaling via survival and mitochondrial reporter assays in two species, where the effect varied between species and tissues. On the other hand, bystander signaling was diminished in worms harvested from a site contaminated with radiation, suggesting that the nature of radiation acting on living organisms must be evaluated on a system-level (Rusin et al. 2019). A recent ex vivo system study by Tuncay Cagatay et al. (2020) showed a variety of cellular changes in recipient bystander mouse embryonic fibroblast (MEF) cells receiving exosomes derived from whole or partial-body organs of mouse irradiation (see later) (Tuncay Cagatay et al. 2020).

Mechanisms of BE

What is currently known of BE mechanisms suggests that IR may affect a much greater volume of tissue than the irradiated volume. Various factors including tissue type, duration of signal exposure, time of analysis, and factors associated with radiation such as type, dose, and dose rate can affect the manifestation of induced BE (Kadhim et al. 2013). Due to the presence of the numerous factors influencing BE, it is difficult to understand their true consequences until we can confirm what is necessary for dictating a response.

As discussed, bystander signal transmission occurs either through gap junctions or secreted factors. Gap junctions permit transfer of small molecular weight molecules and ROS and they act as an integrated unit to mediate BEs and cell population responses, as bystander-induced DNA damage was shown to be abolished in gap junction intercellular communication-deficient cultures (Azzam et al. 2001). Also, a study by Autsavapromporn et al. (2011) demonstrated that gap junctional intercellular communications (GJIC) make a significant contribution to the BE of α particles in terms of propagating toxic effects among high-dose α-particle-irradiated human cells. Our understanding of radiation-induced signaling pathways can be improved by identification of those propagated factors that induce the death of irradiated cells.

A variety of experimental techniques, including media transfer from irradiated cells, co-culture insert systems, partial shielding, and microbeams have been used to demonstrate the role of media borne signals in the extracellular environment (Ghosh et al. 2015). ROS, RNS (Shao et al. 2006), cytokines (Hei et al. 2008) such as IL-8 (Facoetti et al. 2006; Havaki et al. 2015), TGF-beta (Portess et al. 2007), and TNF-alpha (Hamada et al. 2007), calcium fluxes (Lyng et al. 2002), plasma membrane-bound lipid rafts (Hamada et al. 2007), signals associated with the inflammatory response (Lorimore and Wright 2003), and microvesicles, especially exosomes (Albanese and Dainiak 2003; Al-Mayah et al. 2012) have all been demonstrated to play a role in BEs.

Link between BEs and genomic instability

BE and GI are interconnected (Bowler et al. 2006; Ponnaiya et al. 2011; Sawant et al. 2001) and have common phenotypes (Figure 3), including increased mutation and chromosomal rearrangements, micronuclei formation, and up-regulation of oxidative stress; which are expressed at high frequency and with the lack of conventional dose response. For recent reviews see Mothersill et al. (2018) and references within. The link between BE and GI has been experimentally shown using various methods including the grid technique (Lorimore et al. 1998), co-culture in the hemopoietic cell system (Bowler et al. 2006), and low doses of alpha particles (Elbakrawy et al. 2020). The findings from these studies show that the un-irradiated cell population generated majority of their progeny with GI, implying that the damage induced was caused by intercellular communication, i.e. a bystander mechanism. In addition, another study (Sawant et al. 2001) show the increase in oxidative stress in bystander cells is comparable to that seen in radiation-induced GI. The up-regulation of oxidative stress in bystander cells is similar to that observed in radiation-induced GI. Recently, an ex vivo system (Tuncay Cagatay et al. 2020) showed that exosomes derived from organs of whole body irradiated (WBI) or partial body irradiated (PBI) mice induced cellular changes when transferred to naïve MEF cells.

Figure 3. Link between bystander effect and genomic instability.

Extracellular vesicles and exosomes

Extracellular vesicles (EVs) are biological particles that differ by size (<50 nm to several μm in diameter), chemical composition, and function depending on how they are formed and by which cell type they are generated (Théry et al. 2018). EVs have a broad range of physical properties and include exosomes, ectosomes, microvesicles, microparticles, oncosomes, and apoptotic bodies (Doyle and Wang, 2019). As shown in Table 2, the size and presence or absence of specific biochemical markers are the primary characteristics that identify these vesicles (Veziroglu and Mias 2020). EVs have roles associated with an equally broad set of biological processes, including immune response, transfer of functional proteins and nucleic acid, elimination of unwanted materials, nutrition, surface-receptor-mediated cell signaling, and numerous conditions of disease e.g. aging and cancer metastasis.

Table 2. Extracellular vesicle (EV) definitions in the literatures.

Name	Definition	Reference
Microvesicles	Microvesicles are extracellular vesicles that are released from the cell membrane. In multicellular organisms, microvesicles and other EVs vary in size and are found both in tissues and in many types of body fluids	Ratajczak and Ratajczak (2020)
Apoptotic bodies	Apoptotic bodies range in size from 500 to 2000 nm in diameter and are made up of cytoplasm and tightly packed organelles. They are generated as a result of “budding”, a process which consist of extensive plasma membrane blebbing followed by karyorrhexis and disassociation of cell fragments	Elmore (2007) and Stolzing and Grune (2004)
Ectosomes	Ectosomes are shedding membrane vesicles that bud directly from cell membranes of neutrophils or monocytes. They are variable in size from 100 to 1000 nm in diameter, while it is debatable that vesicles larger than 350–400 nm are true ectosomes	Sadallah et al. (2011) and Melodies (2016)
Oncosomes	They are membrane-derived micro and nano scale extracellular vesicles that are released by cancer cells and carry oncogenic messages and protein complexes across cell borders	Meehan et al. (2016)
Exosomes	Exosomes comprise homogenous population of membrane vesicles that range in size between 40 and 100 nm in diameter. They are derived from the endocytic recycling pathway. In endocytosis, endocytic vesicles form at the plasma membrane and fuze to form early endosomes, which then mature into late endosomes where intraluminal vesicles bud off into an intra-cytoplasmic lumen. These multivesicular bodies directly fuze with the plasma membrane and release exosomes into the extracellular space rather than fuzing with lysosome. Instead of fuzing with the lysosome, these multivesicular bodies directly fuze with the plasma membrane and release exosomes into the extracellular space. Exosomal markers used for identification of these vesicles include CD63, CD9 and TSG101	Pant et al. (2012), Mayers and Audhya (2012), Mathivanan et al. (2012) and Record et al. (2011)

Exosomes are a class of EVs secreted by all cell types and have been found in plasma, urine, semen, saliva, cerebral spinal fluid (CSF), bronchial fluid, breast milk, serum, amniotic fluid, synovial fluid, tears, bile, lymph, and gastric acid (review by Doyle and Wang 2019). The discovery of key cargo molecules such as DNA, mRNA, miRNA, lncRNA, protein, metabolites and other components has aroused interest in their function (Valadi et al. 2007; Borges et al. 2013). Although exosomes are produced in the endosomal network, it is difficult to discern between exosomes and plasma membrane-derived vesicles (Kowal et al. 2014). Nevertheless, as compared to conventional plasma membranes, the lipid membrane of EVs has a different composition, with higher levels of sphingomyelin, cholesterol, ceramide, and phosphatidylserine and lower levels of phosphatidylcholine (Laulagnier et al. 2004; Llorente et al. 2013; Kowal et al. 2014).

Exosomes can be internalized by neighboring cells or transported to distant sites via the blood stream. Exosomes are taken up by cells through several processes and released within the recipient cell, while the cargo within has been shown to be functional in the recipient cells by many studies, as extensively reviewed in Zhang et al. (2015), Jelonek et al. (2016), Zhang et al. (2019), Jabbari et al. (2020), Kadhim et al. (2000), Ratajczak and Ratajczak (2020), and Veziroglu and Mias (2020).

Exosomes play a significant role in intercellular communication in the immune system, nervous system, tissue repair, determination of cell phenotype, as well as the progress of carcinogenesis and metastasis (Arscott et al. 2013; Doyle and Wang 2019; Wortzel et al. 2019) and have the potential to modulate paracrine, autocrine and endocrine pathways.

Exosomes and radiation-induced NTE

Exosomes have been linked to cancer, and neurodegenerative diseases (Saeedi et al. 2019), but less is known about their possible role in radiation biology, particularly, radiation-induced NTE. Therefore, in this review, we concentrate on exosomes as delivery vehicles for bystander signals, as well as the downstream effects they have on neighboring cells in relation to radiation-induced NTE. Ionizing radiation induces exosome release in radiation type, dose, time and cell type-dependent manners. This phenomenon occurs as a result of additional stress-inducible pathways of exosome secretion that are activated (Figure 4).

Figure 4. Exosome biogenesis, secretion, and cell communication via exosomes.

Exosome-related research within radiation biology and especially in NTE is a relatively new field, however, there are already existing links. Radiation is a known activator of p53 (O'Hagan and Ljungman 2004), which can regulate exosome release (Yu et al. 2006; Lespagnol et al. 2008; Beer et al. 2015). This shows that exosomes may be regulated by p53 in the presence of cellular stress, such as ionizing radiation. The colocalization of CD29/CD81 complexes increases exosome uptake in response to radiation (Hazawa 2014). However, it is probable that it is the cargo that is important in inducing BEs.

Ionizing radiation has been shown to increase exosome release in many different cell lines (Arscott et al. 2013; Al-Mayah et al. 2015, 2017). Exosome release is also affected by the duration and the dose of irradiation. In a study in which glioblastoma cell line (U87MG) was irradiated with 2, 4, 6, and 8 Gy of X-rays, exosome secretion has been demonstrated to increase in a dose-dependent manner following 24 h after irradiation (Arscott et al. 2013). Moreover, in the same study, PKH26-labeled exosomes derived from irradiated cells were detected on the surface and in the cytoplasm of recipient cells following coincubation of PKH26-labeled exosomes derived from irradiated cells and the recipient cells. This finding indicates that exosomes released from cancer cells can be up taken by cells in the immediate vicinity following irradiation. Another study (Elbakrawy et al. 2020) investigated if radiation can result in senescence through a bystander exosome mechanism. FSF210316B primary human fibroblasts cells were treated with exosomes extracted from irradiated cells’ medium after 0, 0.1, 2, and 10 Gy X-irradiation. Increased levels of senescence were observed when cells were treated with exosomes from irradiated culture medium both at early and delayed time points, being more significant with the latter time point, which demonstrates that delayed senescence is inducible through a bystander mechanism. In a study by Al-Mayah et al. transfer of exosomes derived from 2 Gy X-ray irradiated MCF-7 breast cancer cells to non-irradiated cells caused DNA damage. Exosomes from progeny of irradiated cells had the same effect. Thus, exosomes released by directly irradiated cells and their progeny are significantly involved in mediating both RIBE and the delayed effects of GI (Al-Mayah et al. 2015).

In another study, conditioned media and exosome depleted conditioned media from human keratinocyte cells (HaCaT) irradiated with 0.005, 0.05, and 0.5 Gy γ-rays were transferred to un-irradiated cells. Interestingly, a reduction in cell death, calcium influx, and reactive oxygen species were observed in the exosomes depleted conditioned media recipient cells compared to the cells received conditioned media which indicates that exosomes play a significant role in the ionizing radiation-induced BEs (Jella et al. 2014).

Recently, Tuncay Cagatay et al. investigated the alterations in exosome characteristics and roles of exosomes as putative molecular signaling mediators of radiation damage, using an ex vivo system in which exosomes obtained from whole or partial-body irradiated mice organs were transferred to recipient mouse embryonic fibroblast (MEF) cells following 24 h and 15 days post-irradiation (Tuncay Cagatay et al. 2020). The results show that the number of exosomes is increased in both directly irradiated and abscopal organs in an organ-specific manner, while functional changes are also observed in terms of cell survival, level of DNA damage, calcium, reactive oxygen species and nitric oxide signaling in MEF cells when treated with exosomes from irradiated mice exosomes compared to that of MEF cells treated with exosomes derived from un-irradiated mice. Alterations in exosome profile and BEs observed in this study constitute prominent findings regarding ex vivo systemic effects of early and delayed effects of X-ray irradiation.

Conclusions

Radiation-induced NTE represent an area of radiobiology that to date is not fully understood mechanistically or in terms of implications. It is now known that exosomes are extensively involved in transmitting signals that mediate the RIBE and potentially further link BE with RIGI. The cargo carried within exosomes has the ability to alter epigenetic functions within the cell, and it has been suggested exosomes specifically target epigenetic processes (Zhu et al. 2014). A major component of exosomes is small non-coding RNA in particular miRNA. These have been shown to be functional upon delivery by exosomes, altering gene expression. It has also been observed that exosomes can elicit epigenetic effects through altering the methylation pattern of recipient cells through upregulation of methyltransferases (Qian et al. 2015). This aspect has not been studied extensively in radiation-induced NTE, with both exosomal protein and RNA being implicated in BE at early and delayed time-points. It has also been suggested that a large proportion of exosomal contents are able to alter histones, a key component of DNA structure that has an important role in gene regulation (Sharma 2014). Exosomes are also capable of stimulating inflammatory processes and oxidative stress both of which have been linked to NTE.

In other situations, exosomes have been shown to demonstrate beneficial effects such as anti-inflammatory processes. We are currently unable to discern what confounding factors have the biggest impact on the type of vesicle released; is it cell type, radiation type, dose, or even dose rate? There are, therefore, a number of interesting research questions relating to the whole EV field and not limited to radiation biology. Finding answers to these questions may lead to novel therapies, prognostic and diagnostic biomarkers, and an enhanced understanding of intercellular communication.

Acknowledgments

The authors thank Drs Scott Bright and Ammar Mayah for their help with the figures and Dr Edwin Goodwin for his valuable comments and suggestions on the article.

Disclosure statement

The authors would like to confirm that no relevant review paper is currently under review or in press elsewhere, and also all authors have approved the manuscript and agree to its submission. Additionally, there is no conflict of interest.

Additional information

Funding

The work featured in this article was supported by the HEIF 5 Project Fund from Oxford Brookes University.

Notes on contributors

Munira Kadhim

Munira A. Kadhim, PhD, is a Professor of Radiation Biology and Head of the Genomic Instability and cell communication Research Group in the Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, UK.

Seda Tuncay Cagatay

Seda Tuncay Cagatay, PhD, is a Postdoctoral Research Assistant at the Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, UK.

Eman Mohammed Elbakrawy

Eman Mohammed Elbakrawy, PhD, (previously) Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, UK.